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Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples

Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems. We measured the plasma-mediated viral RNA degradation as well as the plasma- induced loss of viral infectivity using potato virus Y as a model virus due to its confirmed water transmissibility and economic as well as biological importance. We showed that only 1 min of plasma treatment is sufficient for successful inactivation of viruses in water samples with either high or low organic background. The plasma-mediated inactivation was efficient even at markedly higher virus concentrations than those expected in irrigation waters. Obtained results point to reactive oxygen species as the main mode of viral inactivation. Our laboratory-scale experiments conr fi m for the r fi st time that plasma has an excellent potential as the eukaryotic virus inactivation tool for water sources and could thus provide a cost-effective solution for irrigation mediated plant virus transmission. The outstanding inactivation efficiency demonstrated by plasma treatments in water samples offers further expansions of its application to other water sources such as reused wastewater or contaminated drinking waters, as well as other plant, animal, and human waterborne viruses, ultimately leading to the prevention of water scarcity and numerous human, animal, and plant infections worldwide. Keywords Cold atmospheric plasma · Potato virus Y · Virus inactivation · Water decontamination Introduction The availability of clean water is in continued decline due to the increasing global population and food demand, along with higher standards of living and climate change Electronic supplementary material The online version of this (WWAP 2018). Water scarcity has an important impact on article (https ://doi.org/10.1007/s1256 0-019-09388 -y) contains the environment as it affects aquatic organisms, groundwa- supplementary material, which is available to authorized users. ter-dependent terrestrial ecosystems as well as plants and * Arijana Filipić humans (Pfister et al. 2011). Over the past 4 years, water arijana.filipic@nib.si scarcity has been regarded as one of the highest global risks, in terms of its potential impact on humanity (World Eco- Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, nomic Forum 2017). Despite this, 70% of water use world- Slovenia wide goes on account of irrigation (WWAP 2018). This Jožef Stefan International Postgraduate School, Jamova cesta makes agriculture a major environmental burden in terms 39, 1000 Ljubljana, Slovenia of water use (Ridoutt et al. 2018). To tackle this important Department of Surface Engineering and Optoelectronics, global problem, closed irrigation systems that recycle water, Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, such as hydroponic systems, are becoming more common. Slovenia However, such systems can serve as a route for efficient and University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, rapid transmission of pathogens in case of water source Slovenia Vol:.(1234567890) 1 3 Food and Environmental Virology (2019) 11:220–228 221 contamination. Plant pathogens can reduce seed germina- emphasis on the reactive chemical species, CAP has great tion, affect the yield and even destroy entire crops (Syed Ab antimicrobial potential (Guo et al. 2015). The temperature Rahman et al. 2018). of CAP at the point of application is usually < 40 °C, which Water-transmissible viruses are especially problematic, as makes it suitable for treating biological samples (Hoffmann they are usually resistant to wastewater treatment processes et al. 2013). CAP devices for decontamination have been (Carducci et al. 2009) and common disinfection methods tested for various applications, such as in medicine and food that have been developed to target mostly bacteria. Moreo- processing, where they have been shown to be effective ver, viruses can survive in water for long periods of time, can (Scholtz et al. 2015). They have also been used for degrada- be infectious at low doses, and are the source of numerous tion of non-biological (Bansode et al. 2017) and biologi- human, animal and plant infections and epidemics (Mehle cal, mostly bacterial (Rashmei et al. 2016), contaminants and Ravnikar 2012; Shrestha et al. 2018). Potato virus Y in water. Although some studies of CAP–virus interactions (PVY) is a water-transmissible plant virus that can success- have already been performed (for review see Pradeep and fully spread through irrigation systems (Mehle et al. 2014). Chulkyoon 2016) only one brief study has examined the PVY is, economically and scientifically speaking, one of the effects of CAP on a plant virus, tobacco mosaic virus (Han- 10 most important plant viruses worldwide (Scholthof et al. bal et al. 2018), and only one study has described bacte- 2011) and the most important potato viral pathogen which riophage inactivation in water samples by CAP (Guo et al. can cause up to 80% loss in crop production (Kogovšek et al. 2018). NTN 2016). PVY isolates from the recombinant PVY group The aim of the present study was to evaluate the appli- are the most devastating and cause mosaic, chlorotic, and cability of CAP for inactivation of viruses in water from NTN necrotic lesions on leaves as well as necrotic ringspots on closed irrigation systems. We chose PVY as the model tubers (Kogovšek et al. 2016). High losses in potato yield virus because of its demonstrated water transmissibility and pose a big problem since potato is one of the most impor- economic relevance. We showed that CAP can inactivate NTN tant crops in the world (FAO 2018). In addition to potato, high concentrations of PVY in nutrient solution after PVY can also infect other important crops, such as tobacco, only 1 min of treatment and suggested that the inactivation tomato, and pepper (Scholthof et al. 2011). is mainly mediated by the formation of reactive oxygen Removal of viruses from irrigation systems is possible, species. but typically used methods can be expensive [membrane filtration, heating, ultraviolet (UV) light, ozonation], time consuming (slow filtration), require large infrastructure Materials and Methods (slow filtration, heat), frequent maintenance (slow filtration, UV light, ozonation), produce undesirable side components For schematic representation of the experimental design, (chlorination, ozonation), or need additional decontamina- see Fig. 1. tion steps (some types of slow filtration, UV light) (Stew - art-Wade 2011). The greatest weakness of all chemical pro- Virus Source cesses for water decontamination is the generation of toxic by-products as well as production, transport and handling We used two sources of viruses in nutrient solution to create of large amounts of dangerous decontaminants. The main an approximation to water samples of different complexi- limitation of physical methods is that they are effective only ties that represent those used in closed irrigation systems. NTN in water areas that are in the close proximity of the operat- Plants infected with PVY were homogenized and diluted ing device (Kraft 2008). Of all the disinfection methods, in nutrient solution to provide the complex infected water only thermal disinfection has been proven to be suitable for samples (henceforth referred to as ‘infected homogenate’). inactivation of plant viruses in hydroponic production sys- The less complex infected water samples contained only NTN tems (Bandte et al. 2016). Thus it is extremely important purified PVY (Online Resource 1a) diluted in nutrient to develop and implement efficient and environmentally solution. Ultracentrifugation-purified virus particles had friendly approaches for water decontamination that do not lower concentration (henceforth referred to as ‘low con- require toxic chemicals and can be scaled up. One of the centration pure virus’) than chromatography-purified virus technologies that might fulfill these requirements is cold particles (henceforth referred to as ‘high concentration pure atmospheric plasma (CAP). virus’). Untreated samples of infected homogenate and both Plasma is the fourth state of matter and it is generated by types of pure virus were used as positive controls. applying energy to a gas. It is a mixture of charged particles Each infected homogenate was prepared by grinding (i.e., ions, free electrons), reactive species, UV photons, 88 ± 3  mg of the green parts (i.e., leaves and stems) of and neutral particles (i.e., molecules, atoms in the excited potato plants (Solanum tuberosum cv. ‘Pentland Squire’) NTN or ground state). Due to some of these components, with the grown in vitro cultures and infected with PVY . This was 1 3 222 Food and Environmental Virology (2019) 11:220–228 Fig. 1 Schematic representation of the experimental design NTN then mixed with 20 mL nutrient solution that consisted of buffer. PVY was then purified using either a standard tap water with added minerals (Johnson et al. 1994). We purification method that included saccharose and CsCl divided each of the infected homogenates into two samples gradient ultracentrifugation (low concentration pure virus) of 10 mL: one that served as a positive control and was not or convective interaction media (CIM) monolithic chro- treated further, and the other that was treated with hydrogen matography (high concentration pure virus) (Rupar et al. peroxide (H O ) (control treatment, various concentrations) 2013). We added the low concentration pure virus particles 2 2 or CAP (various times). to 10 mL nutrient solution and then either left the sample NTN For isolation of the pure viruses, PVY -infected untreated (positive controls) or treated it with simple mag- tobacco and potato tissues from plants grown in the soil netic stirring (control treatment), gas treatment (control were prepared by grinding them in chilled (to 4 °C) grinding treatment with the gas mixture used for CAP production, 1 3 Food and Environmental Virology (2019) 11:220–228 223 but in the absence of CAP), H O (control treatment, various We performed treatments of the infected homogenates 2 2 concentrations and times), or CAP (various times). The high using CAP for 5, 15, 30, and 45 min, and 1 h in two repeats. concentration pure virus particles stayed untreated (positive Treatments of 2 h and 3 h were performed in a single repeat. control) or underwent CAP treatments (various times). We treated both low and high concentration pure virus using The viral RNA for all of the virus preparations was quan- CAP for 1, 5, and 10 min in a single repeat. tified prior to their treatments using reverse-transcription Optical emission spectroscopy (OES) was used to observe droplet digital PCR (RT-ddPCR). RT-ddPCR was performed the light emitted by the plasma during the treatments. An with One-Step RT-ddPCR advanced kit for probes (Bio-Rad, Avantes AvaSpec-3648 optical spectrometer with a resolu- USA) as described by Mehle et al. (2018) with minor modifi- tion of 0.5 nm from 200 to 1100 nm was used. The inte- cations i.e., thresholds of 2400 were not always used during gration time was set to 1 s. We measured spectra during analyses and data with < 10,000 droplets were not discarded. CAP treatments of various samples: nutrient solution, low concentration pure virus, and infected homogenate. Addi- CAP Source Characterization and Treatment tionally, as a control, the OES spectrum of CAP treatment without sample (CAP in the air) was recorded (Fig. 3). We used a CAP system in the single electrode configuration NTN to investigate the inactivation of PVY in the infected Control Treatments samples (Fig. 2). CAP was created using a mixture of argon (~ 99%) and oxygen (~ 1%), with a constant flow rate of We used a series of control treatments to confirm that any NTN 1 ± 0.2 L/min. The plasma or only the gas mixture was intro- effect on the PVY arose from the CAP treatments. The duced into the infected samples using a perforated quartz first two control treatments consisted of either stirring of glass tube. A copper electrode was inserted in the tube and the low concentration pure virus on the magnetic stirrer for connected to a low-frequency generator (31 kHz) that oper- 1 min or treating it for 1 min with the gas mixture used ated at a peak-to-peak voltage of 6 kV, with total average for CAP production, but in the absence of CAP. The next output power of ~ 3 W. stage control treatment included the addition of H O to 2 2 Fig. 2 Production of cold atmospheric plasma (CAP). a Single elec- part of the panel. The CAP enters the samples in the form of bubbles trode cold atmospheric plasma jet and b its schematic representa- (blurred part of the panel) through four openings, two on each side of tion. c CAP treatment of a sample, during which the plasma stream- the glass tube (Color figure online) ers produced can be seen, as the blue-white structures in the lower 1 3 224 Food and Environmental Virology (2019) 11:220–228 from the pure virus were carried out with QIAmp Viral RNA minikit (Qiagen), according to the manufacturer’s instruc- tions, with minor modifications, namely, luciferase RNA (2 ng/sample) was added to the carrier RNA prior to extrac- tion as an external control and the final elution step was performed with 45 µL of RNAse-free water. Sterile water was used as negative control of the extraction to monitor for potential contaminations during all extractions. After the extractions RNA was amplified for the follow - NTN ing four PVY genes using reverse-transcription polymer- ase chain reaction (RT-PCR): P1 and P3 (which code for proteins involved in virus replication), NIa (which codes for a serine-like cysteine protease), and CP (which codes for the coat protein) (Table 1; Online Resource 1b). This selection Fig. 3 An OES spectra of a submerged CAP during treatment of low of the viral genes enabled us to cover different parts of the concentration pure virus and CAP in the air, in the absence of a sam- viral genome. RT-PCR was prepared using One-Step RT- ple. Vertical lines of the same color represent spectral print of chemi- PCR kit (Qiagen), protocol without Q-solution, according cal species: light blue is for OH, yellow for N , pink for H, dark blue for O, and black for Ar (Color figure online) to the manufacturer`s instructions, with minor modifica- tions, namely, smaller volume reactions were prepared and 5 µL of template RNA was used. The cycling conditions the samples. We applied H O at final concentrations of were 30 min at 50 °C, 15 min at 95 °C, 35 cycles of 30 s at 2 2 12.5 mg/L and 25 mg/L for 15 min with constant stirring 94 °C, 1 min at 52 °C and 1 min at 72 °C, 7 min at 72 °C, for the infected homogenates. For the low concentration and an infinite hold at 4 °C. Sterile water was used as a pure viruses, we applied H O at final concentrations of non-template control of RT-PCR reactions to monitor pos- 2 2 0.5 mg/L and 1 mg/L for 1 min and 25 mg/L for 15 min sible contaminations of the PCR reagents. We detected the with constant stirring. Used H O concentrations reflected amplified PCR products using agarose gel electrophoresis 2 2 those found to be present after various CAP treatments (see and considered RNA as degraded, if at least one of the four Online Resources 2 and 3). targeted genes was not amplified. ReverseT ‑ ranscription PCR Test Plant Infectivity Assay NTN To examine the effects of different treatments on degrada- We used test plant infectivity assays to examine the PVY NTN tion of viral RNA, we first extracted the PVY RNA from infectivity in water samples after the control and CAP treat- samples. RNeasy plant minikit (Qiagen, Germany) was used ments. We mechanically inoculated two leaves of individ- for RNA extractions from infected homogenates according ual tobacco (Nicotiana tabacum, cv. ‘White Burley’) plants to the manufacturer`s instructions, with minor modifications, with nutrient solution (negative control), positive controls namely, without using mercaptoethanol. RNA extractions or treated samples. The inoculation process and growth Table 1 Targeted genes and corresponding oligonucleotide sequences used in RT-PCR Targets Oligonucleotide sequences P1 (codes for protein involved in virus replication) P1_FW: 5′-ATG GCA ACT TAC ACA TCA ACA ATC CAG-3′ P1_R: 5′-TTA TTG AGT AAC CTT GGA ACG TGC ATC A-3′ P3 (codes for protein involved in virus replication) P3_FW: 5′-ATG GGT ATT CCT AAT GCA TGC CCT-3′ P3_R: 5′-TTA CTG GTG TCG CAC ATC ATA TTC TTC C-3′ NIa (codes for a serine-like cysteine protease) NIa_FW: 5′-ATG GCC AAA TCA CTC ATG AGA GGT TTA AG-3′ NIa_R: 5′-TTA TTG CTC TAC AAC AAC ATC ATG ATC AAT TAA ATC C-3′ CP (codes for the coat protein) CP_FW: 5′-ATG GGA AAT GAC ACA ATC GAT-3′ CP_R: 5′-TCA CAT GTT CTT GAC TCC AA-3′ All oligonucleotides were designed within presented study. All oligonucleotides were purchased from Integrated DNA Technologies, USA FW forward oligonucleotides, R reverse oligonucleotides 1 3 Food and Environmental Virology (2019) 11:220–228 225 conditions for the tobacco plants were as described in Mehle in duplicates and all RNA samples were analyzed undiluted et al. (2014). and diluted 10-fold to avoid inhibitory effects. We regularly inspected test plants for development of NTN symptoms of PVY infection (Online Resource 1c), and confirmed viral infectivity and systemic spreading using Results and Discussion reverse-transcription real-time (quantitative) PCR (RT- qPCR). We sampled two developed non-inoculated upper The present study is the first one in the field of the eukar - leaves 14 ± 1 days and 32 ± 1 days post-inoculation, and yotic virus inactivation by CAP for the purpose of water pooled together all of the plant material from the plants inoc- decontamination. Besides examining applicability of CAP ulated with the same sample. We then extracted the RNA for virus inactivation in contaminated water samples, we also with RNeasy plant minikit according to the manufacturer`s investigated the most probable mode of viral inactivation. instructions, with minor modifications i.e., without using Complete loss of virus infectivity (total inactivation) was mercaptoethanol and the final RNA elution was carried achieved in 17 out of 18 CAP treatments (Table 2). Only out with 150 µL of RNAse-free water. After that we per- one repeat of the infected homogenate treated by CAP for NTN formed RT-qPCR using AgPath-ID One-Step RT-qPCR 5 min contained infective PVY , which we detected in the mix (Ambion, USA), as described by Mehle et al. (2014) upper non-inoculated leaves of the test plants. We detected NTN with minor modifications particularly, reactions were run PVY in all of the plants inoculated with the positive Table 2 Different treatments of water samples and their effects on the RNA and the viral infectivity Virus sources Treatment Treatment conditions Viral RNA concentration Viral RNA Viral a b c types (concentration and/or time) (copies/µL of sample) degradation infectivity Infected homogenate H O 12.5 mg/L, 15 min 4.5 × 10 − + 2 2 25 mg/L, 15 min − + d 5 6 e CAP 5 min 7.42 × 10 /1.5 × 10 − +/− d 5 5 e 15 min 7.7 × 10 /4.4 × 10 +/− − d 5 5 30 min 5.6 × 10 /6.5  × 10 + − d 5 6 45 min 4.2 × 10 /1.3 × 10 + − d 6 6 1 h 3.6 × 10 /6.0 × 10 − − 2 h 1.8 × 10 − − 3 h 2.0 × 10 + − f 4 Low concentration pure virus Stirring 1 min 4.0 × 10 − + Gas 1 min − + H O 0.5 mg/L, 1 min − + 2 2 1.0 mg/L, 1 min − + 25 mg/L, 15 min − − CAP 1 min 2.7 × 10 − − 5 min + − 10 min + − g 5 High concentration pure vir us CAP 1 min 2.7 × 10 − − 5 min − − 10 min + – CAP cold atmospheric plasma treatment Viral concentration were determined in positive controls RNA was considered as degraded (+) if at least one of the four targeted genes was not amplified Viruses were considered infective (+) if we detected them with RT-qPCR in upper, non-inoculated leaves of test plants 2 and/or 4 weeks after the inoculation Two repeats of CAP treatments were performed One repeat positive (+), other repeat negative (−) f NTN PVY purified from infected tobacco or potato tissue using a classic purification method that included saccharose and CsCl gradient ultracen- trifugation g NTN PVY purified from infected tobacco or potato tissue using CIM monolithic chromatography 1 3 226 Food and Environmental Virology (2019) 11:220–228 NTN NTN control samples, while we have seen no PVY infections However, the greater PVY inactivation obtained with for plants inoculated with the negative control (nutrient solu- CAP, compared to H O alone, suggests that other plasma 2 2 NTN tion). Minimum time needed for inactivation of viruses in components are also involved in this CAP-mediated PVY infected homogenate was 5 min, whereas only 1 min was inactivation. needed for inactivation of pure virus both in high and low We confirmed this with the OES measurements (Fig.  3) concentration. We speculated that difference in inactivation where we observed increased concentration of OH and O times originated from the amount of organic matter present species that probably served as the precursors for produc- in the infected homogenate, which can absorb the plasma tion of different reactive oxygen species. Ar, O, and H atoms 2 + 2 irradiation and as such might ‘protect’ viruses from it i.e., emission lines and OH emission system (A Σ –X Π) were viruses might become less accessible to the irradiation. The observed for all CAP treatments (Online Resource 5). The most obvious proof that CAP interacts with plant organic presence of OH emission system and Balmer H emission matter was a discoloration of samples in the first few min - line in the OES spectra proves that water vapor is dissoci- utes of treatments (Online Resource 4). Additional cause ated in the plasma. The intensities of spectral features did for the difference could be varied initial amount of virus in not change during the treatments, regardless of the sample the samples, which was determined using RT-ddPCR. Con- type. A new spectral feature, N emission bands, can be seen NTN centrations of PV Y in the infected homogenate ranged only in the OES spectrum of CAP in the air. These are pre- 5 6 from 4.2 × 10 to 6 × 10 RNA copies/µL of sample, while sent due to the diffusion of the ambient air into the plasma for the low and high concentration of pure virus, average stream. The OH is also present in the free air CAP because 4 5 determined concentrations were 3.6 × 10 and 2.7 × 10 RNA of the humidity in the ambient air. However, the OH inten- copies/µL of sample, respectively (Table 2). sity is much smaller compared to the submerged CAP, thus Treated samples were tested for the presence of intact indicating that the water from the samples is evaporated and viral RNA by monitoring four targeted genes. We showed dissociated in the plasma. We did not detect any response NTN that the PVY RNA was successfully degraded by the in the range between 200 and 300 nm, the wavelengths at CAP treatments after 15 min for the infected homogenate, which UV radiation damages nucleotides in different ways and after 5 min for low concentration pure virus (Table 2). (USEPA 2006). That is why we concluded that UV radiation RNA was not degraded in any of the positive controls. Since could not have any impact on the virus inactivation. This RNA was not degraded in all of the experiments in which leaves reactive oxygen species as the crucial CAP compo- infectivity was abolished, it is likely that CAP also damages nents of viral inactivation, an argument supported by various viral coat proteins, which destabilizes the virus particles. research groups (reviewed in Guo et al. 2015). Indeed, coat protein damage alone might be enough to inac- We have performed here a pioneering study using CAP tivate viruses. This is supported by the findings of different treatment for eukaryotic virus inactivation in water samples. research groups which showed that coat protein damage after The use of CAP in our experiments effectively inactivated NTN CAP treatment was a main mode of inactivation of differ - PVY in water samples, both in combination with the ent bacteriophages and mammalian viruses: bacteriophage high organic background from the plant debris (infected NTN lambda and MS2, human adenovirus and feline calicivirus homogenate) and in the pure PVY form. The inactiva- NTN (Aboubakr et al. 2018; Wu et al. 2015; Yasuda et al. 2010; tion was efficient even though the PVY genome con- Zimmermann et al. 2011). centrations were significantly higher than those expected To confirm that the viral inactivation was due to CAP in irrigation waters (Mehle et al. 2014). These new findings treatment, we used control treatments that included H O , confirm the potential that CAP treatments hold in the field 2 2 NTN gas, or stirring. None of them had any effects on the PVY of virus inactivation in irrigation water. They also lay the RNA degradation, for either the infected homogenate groundwork to further studies on other waterborne viruses or the pure virus (Table  2). Moreover, in the infectivity of plant, animal and human origins, and on the opportu- assays, across all of the control treatments, only the high- nities for the scaling up of these CAP treatments. Plasma est H O treatment (25 mg/L) of the pure virus for 15 min systems might also prove useful for decontamination of 2 2 NTN effectively reduced the PVY infectivity. However, the other water sources, such as wastewater, drinking water, NTN same treatment did not affect the PVY infectivity in the and water for recreational use. Implementation of plasma infected homogenate (Table 2). This can be explained by systems in wastewater treatment plants would significantly either higher availability of the organic material (including reduce their running and maintenance costs, and the space viruses) in the infected homogenate with which the H O required (Barillas 2015). That might prove to be an excel- 2 2 can interact or by the presence of the plant enzymes in the lent alternative for many countries that reuse wastewater for infected homogenate that can degrade the H O (Zámocký irrigation without prior disinfection due to economic limita- 2 2 et al. 2012). The data for H O as a control treatment con- tions and the scarcity of fresh water supplies (Moazeni et al. 2 2 firm its implication in plasma-mediated virus inactivation. 2017). Implementation of plasma systems might thus have 1 3 Food and Environmental Virology (2019) 11:220–228 227 plasma and plasma-activated water. Applied and Environmental important positive effects on water quality and might pro- Microbiology, 84(17), 1–10. vide solutions that are greatly needed today. To make this Hanbal, S. E., Takashima, K., Miyashita, S., Ando, S., & Ito, K. (2018). implementation as smooth as possible, additional studies Atmospheric-pressure plasma irradiation can disrupt tobacco are required to define the exact mechanism(s) of action and mosaic virus particles and RNAs to inactivate their infectivity. Archives of Virology, 163(10), 2835–2840. whether CAP treated water can have any effects on human, Hoffmann, C., Berganza, C., & Zhang, J. (2013). Cold Atmospheric animal, and plant cells. plasma: Methods of production and application in dentistry and oncology. Medical Gas Research, 3(1), 21. Acknowledgements This work was financially supported by the Slove- Johnson, J. F., Allan, D. L., & Vance, C. P. (1994). Phosphorus nian Research Agency (Research Core Funding No. P4-0407, Project stress-induced proteoid roots show altered metabolism in Lupi- No. L4-9325 and program for young researchers in the accordance nus albus. Plant Physiology, 104(2), 657–665. with » agreement on (co) financing research activity in 2018 « No. 1000- Kogovšek, P., Pompe-Novak, M., Petek, M., Fragner, L., Weckw- 18-0105), Ministry of Agriculture, Forestry and Food and Domžale- erth, W., & Gruden, K. (2016). Primary metabolism, phenyl- Kamnik Wastewater Treatment Plant.We want to thank Dr. Matevž propanoids and antioxidant pathways are regulated in potato as Rupar for designing the oligonucleotides used in the PCR assay and a response to potato virus Y infection. PLoS ONE, 11(1), 1–20. Dr. Magda Tušek Žnidarič for providing representative transmission Kraft, A. (2008). Electrochemical water disinfection: A short review. NTN electron microscopy micrograph of PVY . The study was performed Platinum Metals Review, 52(3), 177–185. using ddPCR equipment financed by the Metrology Institute of the Mehle, N., Dobnik, D., Ravnikar, M., & Pompe Novak, M. (2018). Republic of Slovenia (MIRS), with financial support from the European Validated reverse transcription droplet digital PCR serves as a Regional Development Fund. This equipment is wholly owned by the higher order method for absolute quantification of Potato virus Republic of Slovenia. Y strains. Analytical and Bioanalytical Chemistry, 410(16), 3815–3825. Mehle, N., Gutiérrez-Aguirre, I., Prezelj, N., Delić, D., Vidic, U., & Compliance with Ethical Standards Ravnikar, M. (2014). Survival and transmission of potato virus Y, pepino mosaic virus, and potato spindle tuber viroid in water. Conflict of interest The authors declare that they have no conflict of Applied and Environmental Microbiology, 80(4), 1455–1462. interest. Mehle, N., & Ravnikar, M. (2012). Plant viruses in aqueous environ- ment—Survival, water mediated transmission and detection. Open Access This article is distributed under the terms of the Crea- Water Research, 46(16), 4902–4917. tive Commons Attribution 4.0 International License (http://creat iveco Moazeni, M., Nikaeen, M., Hadi, M., Moghim, S., Mouhebat, L., mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- Hatamzadeh, M., et al. (2017). Estimation of health risks caused tion, and reproduction in any medium, provided you give appropriate by exposure to enteroviruses from agricultural application of credit to the original author(s) and the source, provide a link to the wastewater effluents. Water Research, 125, 104–113. Creative Commons license, and indicate if changes were made. Pfister, S., Bayer, P., Koehler, A., & Hellweg, S. (2011). Environmen- tal impacts of water use in global crop production: Hotspots and trade-offs with land use. Environmental Science and Technology, 45(13), 5761–5768. 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The United Nations World Water Development Report 2018: Nature-based solutions for water. Paris: WWAP. 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples

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Copyright © 2019 by The Author(s)
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Biomedicine; Virology; Food Science; Chemistry/Food Science, general
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1867-0334
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1867-0342
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10.1007/s12560-019-09388-y
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Abstract

While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems. We measured the plasma-mediated viral RNA degradation as well as the plasma- induced loss of viral infectivity using potato virus Y as a model virus due to its confirmed water transmissibility and economic as well as biological importance. We showed that only 1 min of plasma treatment is sufficient for successful inactivation of viruses in water samples with either high or low organic background. The plasma-mediated inactivation was efficient even at markedly higher virus concentrations than those expected in irrigation waters. Obtained results point to reactive oxygen species as the main mode of viral inactivation. Our laboratory-scale experiments conr fi m for the r fi st time that plasma has an excellent potential as the eukaryotic virus inactivation tool for water sources and could thus provide a cost-effective solution for irrigation mediated plant virus transmission. The outstanding inactivation efficiency demonstrated by plasma treatments in water samples offers further expansions of its application to other water sources such as reused wastewater or contaminated drinking waters, as well as other plant, animal, and human waterborne viruses, ultimately leading to the prevention of water scarcity and numerous human, animal, and plant infections worldwide. Keywords Cold atmospheric plasma · Potato virus Y · Virus inactivation · Water decontamination Introduction The availability of clean water is in continued decline due to the increasing global population and food demand, along with higher standards of living and climate change Electronic supplementary material The online version of this (WWAP 2018). Water scarcity has an important impact on article (https ://doi.org/10.1007/s1256 0-019-09388 -y) contains the environment as it affects aquatic organisms, groundwa- supplementary material, which is available to authorized users. ter-dependent terrestrial ecosystems as well as plants and * Arijana Filipić humans (Pfister et al. 2011). Over the past 4 years, water arijana.filipic@nib.si scarcity has been regarded as one of the highest global risks, in terms of its potential impact on humanity (World Eco- Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, nomic Forum 2017). Despite this, 70% of water use world- Slovenia wide goes on account of irrigation (WWAP 2018). This Jožef Stefan International Postgraduate School, Jamova cesta makes agriculture a major environmental burden in terms 39, 1000 Ljubljana, Slovenia of water use (Ridoutt et al. 2018). To tackle this important Department of Surface Engineering and Optoelectronics, global problem, closed irrigation systems that recycle water, Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, such as hydroponic systems, are becoming more common. Slovenia However, such systems can serve as a route for efficient and University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, rapid transmission of pathogens in case of water source Slovenia Vol:.(1234567890) 1 3 Food and Environmental Virology (2019) 11:220–228 221 contamination. Plant pathogens can reduce seed germina- emphasis on the reactive chemical species, CAP has great tion, affect the yield and even destroy entire crops (Syed Ab antimicrobial potential (Guo et al. 2015). The temperature Rahman et al. 2018). of CAP at the point of application is usually < 40 °C, which Water-transmissible viruses are especially problematic, as makes it suitable for treating biological samples (Hoffmann they are usually resistant to wastewater treatment processes et al. 2013). CAP devices for decontamination have been (Carducci et al. 2009) and common disinfection methods tested for various applications, such as in medicine and food that have been developed to target mostly bacteria. Moreo- processing, where they have been shown to be effective ver, viruses can survive in water for long periods of time, can (Scholtz et al. 2015). They have also been used for degrada- be infectious at low doses, and are the source of numerous tion of non-biological (Bansode et al. 2017) and biologi- human, animal and plant infections and epidemics (Mehle cal, mostly bacterial (Rashmei et al. 2016), contaminants and Ravnikar 2012; Shrestha et al. 2018). Potato virus Y in water. Although some studies of CAP–virus interactions (PVY) is a water-transmissible plant virus that can success- have already been performed (for review see Pradeep and fully spread through irrigation systems (Mehle et al. 2014). Chulkyoon 2016) only one brief study has examined the PVY is, economically and scientifically speaking, one of the effects of CAP on a plant virus, tobacco mosaic virus (Han- 10 most important plant viruses worldwide (Scholthof et al. bal et al. 2018), and only one study has described bacte- 2011) and the most important potato viral pathogen which riophage inactivation in water samples by CAP (Guo et al. can cause up to 80% loss in crop production (Kogovšek et al. 2018). NTN 2016). PVY isolates from the recombinant PVY group The aim of the present study was to evaluate the appli- are the most devastating and cause mosaic, chlorotic, and cability of CAP for inactivation of viruses in water from NTN necrotic lesions on leaves as well as necrotic ringspots on closed irrigation systems. We chose PVY as the model tubers (Kogovšek et al. 2016). High losses in potato yield virus because of its demonstrated water transmissibility and pose a big problem since potato is one of the most impor- economic relevance. We showed that CAP can inactivate NTN tant crops in the world (FAO 2018). In addition to potato, high concentrations of PVY in nutrient solution after PVY can also infect other important crops, such as tobacco, only 1 min of treatment and suggested that the inactivation tomato, and pepper (Scholthof et al. 2011). is mainly mediated by the formation of reactive oxygen Removal of viruses from irrigation systems is possible, species. but typically used methods can be expensive [membrane filtration, heating, ultraviolet (UV) light, ozonation], time consuming (slow filtration), require large infrastructure Materials and Methods (slow filtration, heat), frequent maintenance (slow filtration, UV light, ozonation), produce undesirable side components For schematic representation of the experimental design, (chlorination, ozonation), or need additional decontamina- see Fig. 1. tion steps (some types of slow filtration, UV light) (Stew - art-Wade 2011). The greatest weakness of all chemical pro- Virus Source cesses for water decontamination is the generation of toxic by-products as well as production, transport and handling We used two sources of viruses in nutrient solution to create of large amounts of dangerous decontaminants. The main an approximation to water samples of different complexi- limitation of physical methods is that they are effective only ties that represent those used in closed irrigation systems. NTN in water areas that are in the close proximity of the operat- Plants infected with PVY were homogenized and diluted ing device (Kraft 2008). Of all the disinfection methods, in nutrient solution to provide the complex infected water only thermal disinfection has been proven to be suitable for samples (henceforth referred to as ‘infected homogenate’). inactivation of plant viruses in hydroponic production sys- The less complex infected water samples contained only NTN tems (Bandte et al. 2016). Thus it is extremely important purified PVY (Online Resource 1a) diluted in nutrient to develop and implement efficient and environmentally solution. Ultracentrifugation-purified virus particles had friendly approaches for water decontamination that do not lower concentration (henceforth referred to as ‘low con- require toxic chemicals and can be scaled up. One of the centration pure virus’) than chromatography-purified virus technologies that might fulfill these requirements is cold particles (henceforth referred to as ‘high concentration pure atmospheric plasma (CAP). virus’). Untreated samples of infected homogenate and both Plasma is the fourth state of matter and it is generated by types of pure virus were used as positive controls. applying energy to a gas. It is a mixture of charged particles Each infected homogenate was prepared by grinding (i.e., ions, free electrons), reactive species, UV photons, 88 ± 3  mg of the green parts (i.e., leaves and stems) of and neutral particles (i.e., molecules, atoms in the excited potato plants (Solanum tuberosum cv. ‘Pentland Squire’) NTN or ground state). Due to some of these components, with the grown in vitro cultures and infected with PVY . This was 1 3 222 Food and Environmental Virology (2019) 11:220–228 Fig. 1 Schematic representation of the experimental design NTN then mixed with 20 mL nutrient solution that consisted of buffer. PVY was then purified using either a standard tap water with added minerals (Johnson et al. 1994). We purification method that included saccharose and CsCl divided each of the infected homogenates into two samples gradient ultracentrifugation (low concentration pure virus) of 10 mL: one that served as a positive control and was not or convective interaction media (CIM) monolithic chro- treated further, and the other that was treated with hydrogen matography (high concentration pure virus) (Rupar et al. peroxide (H O ) (control treatment, various concentrations) 2013). We added the low concentration pure virus particles 2 2 or CAP (various times). to 10 mL nutrient solution and then either left the sample NTN For isolation of the pure viruses, PVY -infected untreated (positive controls) or treated it with simple mag- tobacco and potato tissues from plants grown in the soil netic stirring (control treatment), gas treatment (control were prepared by grinding them in chilled (to 4 °C) grinding treatment with the gas mixture used for CAP production, 1 3 Food and Environmental Virology (2019) 11:220–228 223 but in the absence of CAP), H O (control treatment, various We performed treatments of the infected homogenates 2 2 concentrations and times), or CAP (various times). The high using CAP for 5, 15, 30, and 45 min, and 1 h in two repeats. concentration pure virus particles stayed untreated (positive Treatments of 2 h and 3 h were performed in a single repeat. control) or underwent CAP treatments (various times). We treated both low and high concentration pure virus using The viral RNA for all of the virus preparations was quan- CAP for 1, 5, and 10 min in a single repeat. tified prior to their treatments using reverse-transcription Optical emission spectroscopy (OES) was used to observe droplet digital PCR (RT-ddPCR). RT-ddPCR was performed the light emitted by the plasma during the treatments. An with One-Step RT-ddPCR advanced kit for probes (Bio-Rad, Avantes AvaSpec-3648 optical spectrometer with a resolu- USA) as described by Mehle et al. (2018) with minor modifi- tion of 0.5 nm from 200 to 1100 nm was used. The inte- cations i.e., thresholds of 2400 were not always used during gration time was set to 1 s. We measured spectra during analyses and data with < 10,000 droplets were not discarded. CAP treatments of various samples: nutrient solution, low concentration pure virus, and infected homogenate. Addi- CAP Source Characterization and Treatment tionally, as a control, the OES spectrum of CAP treatment without sample (CAP in the air) was recorded (Fig. 3). We used a CAP system in the single electrode configuration NTN to investigate the inactivation of PVY in the infected Control Treatments samples (Fig. 2). CAP was created using a mixture of argon (~ 99%) and oxygen (~ 1%), with a constant flow rate of We used a series of control treatments to confirm that any NTN 1 ± 0.2 L/min. The plasma or only the gas mixture was intro- effect on the PVY arose from the CAP treatments. The duced into the infected samples using a perforated quartz first two control treatments consisted of either stirring of glass tube. A copper electrode was inserted in the tube and the low concentration pure virus on the magnetic stirrer for connected to a low-frequency generator (31 kHz) that oper- 1 min or treating it for 1 min with the gas mixture used ated at a peak-to-peak voltage of 6 kV, with total average for CAP production, but in the absence of CAP. The next output power of ~ 3 W. stage control treatment included the addition of H O to 2 2 Fig. 2 Production of cold atmospheric plasma (CAP). a Single elec- part of the panel. The CAP enters the samples in the form of bubbles trode cold atmospheric plasma jet and b its schematic representa- (blurred part of the panel) through four openings, two on each side of tion. c CAP treatment of a sample, during which the plasma stream- the glass tube (Color figure online) ers produced can be seen, as the blue-white structures in the lower 1 3 224 Food and Environmental Virology (2019) 11:220–228 from the pure virus were carried out with QIAmp Viral RNA minikit (Qiagen), according to the manufacturer’s instruc- tions, with minor modifications, namely, luciferase RNA (2 ng/sample) was added to the carrier RNA prior to extrac- tion as an external control and the final elution step was performed with 45 µL of RNAse-free water. Sterile water was used as negative control of the extraction to monitor for potential contaminations during all extractions. After the extractions RNA was amplified for the follow - NTN ing four PVY genes using reverse-transcription polymer- ase chain reaction (RT-PCR): P1 and P3 (which code for proteins involved in virus replication), NIa (which codes for a serine-like cysteine protease), and CP (which codes for the coat protein) (Table 1; Online Resource 1b). This selection Fig. 3 An OES spectra of a submerged CAP during treatment of low of the viral genes enabled us to cover different parts of the concentration pure virus and CAP in the air, in the absence of a sam- viral genome. RT-PCR was prepared using One-Step RT- ple. Vertical lines of the same color represent spectral print of chemi- PCR kit (Qiagen), protocol without Q-solution, according cal species: light blue is for OH, yellow for N , pink for H, dark blue for O, and black for Ar (Color figure online) to the manufacturer`s instructions, with minor modifica- tions, namely, smaller volume reactions were prepared and 5 µL of template RNA was used. The cycling conditions the samples. We applied H O at final concentrations of were 30 min at 50 °C, 15 min at 95 °C, 35 cycles of 30 s at 2 2 12.5 mg/L and 25 mg/L for 15 min with constant stirring 94 °C, 1 min at 52 °C and 1 min at 72 °C, 7 min at 72 °C, for the infected homogenates. For the low concentration and an infinite hold at 4 °C. Sterile water was used as a pure viruses, we applied H O at final concentrations of non-template control of RT-PCR reactions to monitor pos- 2 2 0.5 mg/L and 1 mg/L for 1 min and 25 mg/L for 15 min sible contaminations of the PCR reagents. We detected the with constant stirring. Used H O concentrations reflected amplified PCR products using agarose gel electrophoresis 2 2 those found to be present after various CAP treatments (see and considered RNA as degraded, if at least one of the four Online Resources 2 and 3). targeted genes was not amplified. ReverseT ‑ ranscription PCR Test Plant Infectivity Assay NTN To examine the effects of different treatments on degrada- We used test plant infectivity assays to examine the PVY NTN tion of viral RNA, we first extracted the PVY RNA from infectivity in water samples after the control and CAP treat- samples. RNeasy plant minikit (Qiagen, Germany) was used ments. We mechanically inoculated two leaves of individ- for RNA extractions from infected homogenates according ual tobacco (Nicotiana tabacum, cv. ‘White Burley’) plants to the manufacturer`s instructions, with minor modifications, with nutrient solution (negative control), positive controls namely, without using mercaptoethanol. RNA extractions or treated samples. The inoculation process and growth Table 1 Targeted genes and corresponding oligonucleotide sequences used in RT-PCR Targets Oligonucleotide sequences P1 (codes for protein involved in virus replication) P1_FW: 5′-ATG GCA ACT TAC ACA TCA ACA ATC CAG-3′ P1_R: 5′-TTA TTG AGT AAC CTT GGA ACG TGC ATC A-3′ P3 (codes for protein involved in virus replication) P3_FW: 5′-ATG GGT ATT CCT AAT GCA TGC CCT-3′ P3_R: 5′-TTA CTG GTG TCG CAC ATC ATA TTC TTC C-3′ NIa (codes for a serine-like cysteine protease) NIa_FW: 5′-ATG GCC AAA TCA CTC ATG AGA GGT TTA AG-3′ NIa_R: 5′-TTA TTG CTC TAC AAC AAC ATC ATG ATC AAT TAA ATC C-3′ CP (codes for the coat protein) CP_FW: 5′-ATG GGA AAT GAC ACA ATC GAT-3′ CP_R: 5′-TCA CAT GTT CTT GAC TCC AA-3′ All oligonucleotides were designed within presented study. All oligonucleotides were purchased from Integrated DNA Technologies, USA FW forward oligonucleotides, R reverse oligonucleotides 1 3 Food and Environmental Virology (2019) 11:220–228 225 conditions for the tobacco plants were as described in Mehle in duplicates and all RNA samples were analyzed undiluted et al. (2014). and diluted 10-fold to avoid inhibitory effects. We regularly inspected test plants for development of NTN symptoms of PVY infection (Online Resource 1c), and confirmed viral infectivity and systemic spreading using Results and Discussion reverse-transcription real-time (quantitative) PCR (RT- qPCR). We sampled two developed non-inoculated upper The present study is the first one in the field of the eukar - leaves 14 ± 1 days and 32 ± 1 days post-inoculation, and yotic virus inactivation by CAP for the purpose of water pooled together all of the plant material from the plants inoc- decontamination. Besides examining applicability of CAP ulated with the same sample. We then extracted the RNA for virus inactivation in contaminated water samples, we also with RNeasy plant minikit according to the manufacturer`s investigated the most probable mode of viral inactivation. instructions, with minor modifications i.e., without using Complete loss of virus infectivity (total inactivation) was mercaptoethanol and the final RNA elution was carried achieved in 17 out of 18 CAP treatments (Table 2). Only out with 150 µL of RNAse-free water. After that we per- one repeat of the infected homogenate treated by CAP for NTN formed RT-qPCR using AgPath-ID One-Step RT-qPCR 5 min contained infective PVY , which we detected in the mix (Ambion, USA), as described by Mehle et al. (2014) upper non-inoculated leaves of the test plants. We detected NTN with minor modifications particularly, reactions were run PVY in all of the plants inoculated with the positive Table 2 Different treatments of water samples and their effects on the RNA and the viral infectivity Virus sources Treatment Treatment conditions Viral RNA concentration Viral RNA Viral a b c types (concentration and/or time) (copies/µL of sample) degradation infectivity Infected homogenate H O 12.5 mg/L, 15 min 4.5 × 10 − + 2 2 25 mg/L, 15 min − + d 5 6 e CAP 5 min 7.42 × 10 /1.5 × 10 − +/− d 5 5 e 15 min 7.7 × 10 /4.4 × 10 +/− − d 5 5 30 min 5.6 × 10 /6.5  × 10 + − d 5 6 45 min 4.2 × 10 /1.3 × 10 + − d 6 6 1 h 3.6 × 10 /6.0 × 10 − − 2 h 1.8 × 10 − − 3 h 2.0 × 10 + − f 4 Low concentration pure virus Stirring 1 min 4.0 × 10 − + Gas 1 min − + H O 0.5 mg/L, 1 min − + 2 2 1.0 mg/L, 1 min − + 25 mg/L, 15 min − − CAP 1 min 2.7 × 10 − − 5 min + − 10 min + − g 5 High concentration pure vir us CAP 1 min 2.7 × 10 − − 5 min − − 10 min + – CAP cold atmospheric plasma treatment Viral concentration were determined in positive controls RNA was considered as degraded (+) if at least one of the four targeted genes was not amplified Viruses were considered infective (+) if we detected them with RT-qPCR in upper, non-inoculated leaves of test plants 2 and/or 4 weeks after the inoculation Two repeats of CAP treatments were performed One repeat positive (+), other repeat negative (−) f NTN PVY purified from infected tobacco or potato tissue using a classic purification method that included saccharose and CsCl gradient ultracen- trifugation g NTN PVY purified from infected tobacco or potato tissue using CIM monolithic chromatography 1 3 226 Food and Environmental Virology (2019) 11:220–228 NTN NTN control samples, while we have seen no PVY infections However, the greater PVY inactivation obtained with for plants inoculated with the negative control (nutrient solu- CAP, compared to H O alone, suggests that other plasma 2 2 NTN tion). Minimum time needed for inactivation of viruses in components are also involved in this CAP-mediated PVY infected homogenate was 5 min, whereas only 1 min was inactivation. needed for inactivation of pure virus both in high and low We confirmed this with the OES measurements (Fig.  3) concentration. We speculated that difference in inactivation where we observed increased concentration of OH and O times originated from the amount of organic matter present species that probably served as the precursors for produc- in the infected homogenate, which can absorb the plasma tion of different reactive oxygen species. Ar, O, and H atoms 2 + 2 irradiation and as such might ‘protect’ viruses from it i.e., emission lines and OH emission system (A Σ –X Π) were viruses might become less accessible to the irradiation. The observed for all CAP treatments (Online Resource 5). The most obvious proof that CAP interacts with plant organic presence of OH emission system and Balmer H emission matter was a discoloration of samples in the first few min - line in the OES spectra proves that water vapor is dissoci- utes of treatments (Online Resource 4). Additional cause ated in the plasma. The intensities of spectral features did for the difference could be varied initial amount of virus in not change during the treatments, regardless of the sample the samples, which was determined using RT-ddPCR. Con- type. A new spectral feature, N emission bands, can be seen NTN centrations of PV Y in the infected homogenate ranged only in the OES spectrum of CAP in the air. These are pre- 5 6 from 4.2 × 10 to 6 × 10 RNA copies/µL of sample, while sent due to the diffusion of the ambient air into the plasma for the low and high concentration of pure virus, average stream. The OH is also present in the free air CAP because 4 5 determined concentrations were 3.6 × 10 and 2.7 × 10 RNA of the humidity in the ambient air. However, the OH inten- copies/µL of sample, respectively (Table 2). sity is much smaller compared to the submerged CAP, thus Treated samples were tested for the presence of intact indicating that the water from the samples is evaporated and viral RNA by monitoring four targeted genes. We showed dissociated in the plasma. We did not detect any response NTN that the PVY RNA was successfully degraded by the in the range between 200 and 300 nm, the wavelengths at CAP treatments after 15 min for the infected homogenate, which UV radiation damages nucleotides in different ways and after 5 min for low concentration pure virus (Table 2). (USEPA 2006). That is why we concluded that UV radiation RNA was not degraded in any of the positive controls. Since could not have any impact on the virus inactivation. This RNA was not degraded in all of the experiments in which leaves reactive oxygen species as the crucial CAP compo- infectivity was abolished, it is likely that CAP also damages nents of viral inactivation, an argument supported by various viral coat proteins, which destabilizes the virus particles. research groups (reviewed in Guo et al. 2015). Indeed, coat protein damage alone might be enough to inac- We have performed here a pioneering study using CAP tivate viruses. This is supported by the findings of different treatment for eukaryotic virus inactivation in water samples. research groups which showed that coat protein damage after The use of CAP in our experiments effectively inactivated NTN CAP treatment was a main mode of inactivation of differ - PVY in water samples, both in combination with the ent bacteriophages and mammalian viruses: bacteriophage high organic background from the plant debris (infected NTN lambda and MS2, human adenovirus and feline calicivirus homogenate) and in the pure PVY form. The inactiva- NTN (Aboubakr et al. 2018; Wu et al. 2015; Yasuda et al. 2010; tion was efficient even though the PVY genome con- Zimmermann et al. 2011). centrations were significantly higher than those expected To confirm that the viral inactivation was due to CAP in irrigation waters (Mehle et al. 2014). These new findings treatment, we used control treatments that included H O , confirm the potential that CAP treatments hold in the field 2 2 NTN gas, or stirring. None of them had any effects on the PVY of virus inactivation in irrigation water. They also lay the RNA degradation, for either the infected homogenate groundwork to further studies on other waterborne viruses or the pure virus (Table  2). Moreover, in the infectivity of plant, animal and human origins, and on the opportu- assays, across all of the control treatments, only the high- nities for the scaling up of these CAP treatments. Plasma est H O treatment (25 mg/L) of the pure virus for 15 min systems might also prove useful for decontamination of 2 2 NTN effectively reduced the PVY infectivity. However, the other water sources, such as wastewater, drinking water, NTN same treatment did not affect the PVY infectivity in the and water for recreational use. Implementation of plasma infected homogenate (Table 2). This can be explained by systems in wastewater treatment plants would significantly either higher availability of the organic material (including reduce their running and maintenance costs, and the space viruses) in the infected homogenate with which the H O required (Barillas 2015). That might prove to be an excel- 2 2 can interact or by the presence of the plant enzymes in the lent alternative for many countries that reuse wastewater for infected homogenate that can degrade the H O (Zámocký irrigation without prior disinfection due to economic limita- 2 2 et al. 2012). The data for H O as a control treatment con- tions and the scarcity of fresh water supplies (Moazeni et al. 2 2 firm its implication in plasma-mediated virus inactivation. 2017). Implementation of plasma systems might thus have 1 3 Food and Environmental Virology (2019) 11:220–228 227 plasma and plasma-activated water. Applied and Environmental important positive effects on water quality and might pro- Microbiology, 84(17), 1–10. vide solutions that are greatly needed today. To make this Hanbal, S. E., Takashima, K., Miyashita, S., Ando, S., & Ito, K. (2018). implementation as smooth as possible, additional studies Atmospheric-pressure plasma irradiation can disrupt tobacco are required to define the exact mechanism(s) of action and mosaic virus particles and RNAs to inactivate their infectivity. Archives of Virology, 163(10), 2835–2840. whether CAP treated water can have any effects on human, Hoffmann, C., Berganza, C., & Zhang, J. (2013). Cold Atmospheric animal, and plant cells. plasma: Methods of production and application in dentistry and oncology. Medical Gas Research, 3(1), 21. Acknowledgements This work was financially supported by the Slove- Johnson, J. F., Allan, D. L., & Vance, C. P. (1994). Phosphorus nian Research Agency (Research Core Funding No. P4-0407, Project stress-induced proteoid roots show altered metabolism in Lupi- No. L4-9325 and program for young researchers in the accordance nus albus. Plant Physiology, 104(2), 657–665. with » agreement on (co) financing research activity in 2018 « No. 1000- Kogovšek, P., Pompe-Novak, M., Petek, M., Fragner, L., Weckw- 18-0105), Ministry of Agriculture, Forestry and Food and Domžale- erth, W., & Gruden, K. (2016). Primary metabolism, phenyl- Kamnik Wastewater Treatment Plant.We want to thank Dr. Matevž propanoids and antioxidant pathways are regulated in potato as Rupar for designing the oligonucleotides used in the PCR assay and a response to potato virus Y infection. PLoS ONE, 11(1), 1–20. Dr. Magda Tušek Žnidarič for providing representative transmission Kraft, A. (2008). Electrochemical water disinfection: A short review. NTN electron microscopy micrograph of PVY . The study was performed Platinum Metals Review, 52(3), 177–185. using ddPCR equipment financed by the Metrology Institute of the Mehle, N., Dobnik, D., Ravnikar, M., & Pompe Novak, M. (2018). Republic of Slovenia (MIRS), with financial support from the European Validated reverse transcription droplet digital PCR serves as a Regional Development Fund. This equipment is wholly owned by the higher order method for absolute quantification of Potato virus Republic of Slovenia. Y strains. Analytical and Bioanalytical Chemistry, 410(16), 3815–3825. Mehle, N., Gutiérrez-Aguirre, I., Prezelj, N., Delić, D., Vidic, U., & Compliance with Ethical Standards Ravnikar, M. (2014). Survival and transmission of potato virus Y, pepino mosaic virus, and potato spindle tuber viroid in water. Conflict of interest The authors declare that they have no conflict of Applied and Environmental Microbiology, 80(4), 1455–1462. interest. Mehle, N., & Ravnikar, M. (2012). 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The United Nations World Water Development Report 2018: Nature-based solutions for water. Paris: WWAP. 1 3

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